During mitosis the mitotic spindle communicates essential information to the cell cortex to determine the time and place of cell cleavage. We intend to address the signaling mechanism underlying this fundamental process. Recent analysis in mammalian cells has demonstrated a potential role for passenger proteins such as Survivin, Aurora B, INCENP and TD-60 in this process. Passenger proteins are proteins that migrate from the kinetochores to the spindle equator during late mitosis and have a probable function in signaling the time and position of cleavage furrow formation. Further, three microtubule associated proteins, PRC1, MKLP1 and Cyk-4, are necessary for organization of the midzone spindle. We have done substantial recent work on the roles of Survivin and PRC1 in cell cleavage. We have determined the X-ray structure of Survivin and have demonstrated that it is homodimeric with two extended alpha-helical tails. We have further determined that Survivin is a passenger protein. In a parallel project, we have determined that PRC1 is a microtubule binding protein that is required for bundling of microtubules in the spindle midzone. In the absence of PRC1, depleted by siRNA transfection, the midzone spindle has no organization and cells are incapable of proceeding with cleavage. The primary focus of this application is to conduct a domain analysis of PRC1 and of Survivin in order to understand how the central spindle is constructed in anaphase, to define what role the central spindle plays in organizing the passenger proteins, and to understand the interplay of passenger proteins with the central spindle in the process of signaling cell cleavage.